U.S. patent application number 15/302780 was filed with the patent office on 2017-02-02 for method and device for two-dimensional separation of ionic species.
The applicant listed for this patent is UNIVERSITAT REGENSBURG. Invention is credited to Andrea BEUTNER, Sven KOCHMANN, Jonas MARK, Frank-Michael MATYSIK.
Application Number | 20170030860 15/302780 |
Document ID | / |
Family ID | 53039880 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170030860 |
Kind Code |
A1 |
MATYSIK; Frank-Michael ; et
al. |
February 2, 2017 |
METHOD AND DEVICE FOR TWO-DIMENSIONAL SEPARATION OF IONIC
SPECIES
Abstract
The invention relates to a method which realizes a
two-dimensional separation of ionic species on the basis of the
online coupling of ion chromatography (IC) and capillary
electrophoresis (CE). A device for IC.times.CE coupling, its
implementation in terms of two alternatives, the connection to a
mass spectrometric detector, and corresponding application are
described.
Inventors: |
MATYSIK; Frank-Michael;
(Regensburg, DE) ; BEUTNER; Andrea; (Etzenricht,
DE) ; MARK; Jonas; (Kohlberg, DE) ; KOCHMANN;
Sven; (Regensburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITAT REGENSBURG |
Regensburg |
|
DE |
|
|
Family ID: |
53039880 |
Appl. No.: |
15/302780 |
Filed: |
April 23, 2015 |
PCT Filed: |
April 23, 2015 |
PCT NO: |
PCT/EP2015/058837 |
371 Date: |
October 7, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 30/465 20130101;
G01N 30/96 20130101; B01D 15/361 20130101; G01N 2030/204 20130101;
G01N 27/44743 20130101; G01N 30/20 20130101; G01N 2030/965
20130101; G01N 30/16 20130101; G01N 27/44782 20130101; G01N 30/463
20130101; G01N 27/44791 20130101 |
International
Class: |
G01N 27/447 20060101
G01N027/447; G01N 30/20 20060101 G01N030/20; G01N 30/16 20060101
G01N030/16; B01D 15/36 20060101 B01D015/36; G01N 30/96 20060101
G01N030/96 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2014 |
EP |
14166119.9 |
Feb 26, 2015 |
EP |
15156828.4 |
Claims
1. Device for continuous two-dimensional separation of ionic
species comprising a) an ion chromatography (IC) system, comprising
a suppressor; b) a capillary electrophoresis (CE) system comprising
an electrolyte vessel and a high voltage electrode; and c) a
modulator, for transferring effluent of the IC system to the CE
system, comprising a transfer capillary and injector means, wherein
the injector means are adapted to provide discrete volume segments
of effluent.
2. The device of claim 1, wherein the injector means comprises a
positioning and guidance system for modifying the distance between
the outlet of the transfer capillary and the inlet of a separation
capillary in controlled manner.
3. The device of claim 1, wherein the injector means comprises a
switching valve between transfer capillary and separation capillary
for controlling and guiding volume segments of the effluent to the
inlet of the separation capillary.
4. The device of claim 1, wherein the injector means comprises a
microprocessor for controlling the provision and/or delivery of
volume segments.
5. The device of claim 1, wherein the CE separation capillary is a
short capillary electrophoresis (CE) separation capillary, which is
less than 50 cm in length, and has an inner diameter of less than
100 .mu.m, wherein the inlet of the separation capillary is in
alignment with the outlet of the transfer capillary.
6. Device according to claim 1, further comprising a detector
connected to the outlet of the separation capillary, wherein
optionally the detector is a mass spectrometer.
7. Method for two-dimensional separation of ionic species by online
coupling of ion chromatography (IC) and capillary electrophoresis
(CE), comprising the following steps: a) injecting a sample into an
IC system comprising a suppressor; b) transferring IC effluent
through a transfer capillary to a CE system comprising an
electrolyte vessel with electrophoresis buffer, a separation
capillary and a high voltage electrode; and c) injecting volume
segments of effluent to a separation capillary of the CE system via
injector means.
8. The method of claim 7, wherein the distance between the outlet
of the transfer capillary and the inlet of a separation capillary
of a CE system is periodically modified between a first position
and a second position by movement of one or both capillaries,
wherein the first position provides for a distance of more than 150
.mu.m and the second position provides for a distance of less than
100 .mu.m, wherein the movement is controlled by a modulator
comprising a positioning and guidance system.
9. The method of claim 8, wherein both capillaries in the second
position are in alignment and/or wherein the movement of one or
both capillaries is in axial direction.
10. The method of claim 8, wherein the short distance is kept for
less than 10 seconds, wherein during this step a volume segment is
introduced into the inlet of the separation capillary of the CE
system.
11. The method of claim 7, wherein the IC system is a capillary
system, and wherein the flow rate in the capillary IC system is
less than 10 .mu.l/min, preferably about 1 to about 5
.mu.l/min.
12. Method of claim 7, wherein conductivity of the IC carrier flow
is detected.
13. Method of claim 7, wherein the detector is connected to the
outlet of the separation capillary, wherein optionally the detector
is a mass spectrometer, coupled via a sheath flow electrospray
ionization (ESI) interface.
14. Method of claim 7, wherein a short distance between outlet of
the transfer capillary and inlet of the separation capillary is
kept for less than 2 seconds, and/or wherein the short distance
between the outlet of the transfer capillary and the inlet of a
separation capillary of a CE system is between 10 and 50 .mu.m;
and/or wherein the increased distance between the outlet of the
transfer capillary and the inlet of a separation capillary of a CE
system is about 200 to 350 .mu.m.
15. Method of claim 7, wherein the separation capillary is in a
fixed position, and the movement of the transfer capillary is
controlled by the positioning and guidance system
Description
FIELD OF THE INVENTION
[0001] The invention is concerned with a method and a device for
two-dimensional separation of ionic species using ion
chromatography (IC) and capillary electrophoresis (CE).
BACKGROUND
[0002] In the last decades a plurality of analytical methods using
chromatographic technology has been provided. Further refined
methods have been developed by coupling two chromatographic
techniques. An example is comprehensive two-dimensional gas
chromatography (GC.times.GC), in which usually a long nonpolar GC
column is coupled with a short polar GC column, with the aim to
improve the so-called peak capacity, which is a performance measure
describing the number of resolved signals of the analytical
separation in a specified time slot. The separation on the long
column leads to typical retention times in the minute range, while
fast separations with orthogonal selectivity can be realized in the
second range on the short column. The results of the GC.times.GC
technique can be presented as so-called contour plots, wherein
signal intensities are assigned to the retention times of the first
and second separation dimension on the basis of a color scale.
[0003] In analogy to the GC.times.GC, other two-dimensional
chromatography systems have been described for separations with
liquid mobile phases. Two-dimensional liquid chromatography
(LC.times.LC) has found increased use [1]. Recently, a combination
of liquid chromatography (LC) and chip electrophoresis has been
described [2]. Also known are two-dimensional separations of
various electrophoretic separation methods as well as the coupling
of ion chromatography (IC) and reversed phase liquid chromatography
[3].
[0004] For the separation of ionic species ion chromatography (IC)
and capillary electrophoresis (CE) are the major instrumental
techniques. Both separation methods are based on completely
different separation mechanisms.
[0005] Ion chromatography is a method that allows the separation of
ions and protonated/deprotonated polar molecules based on their
affinity to an ion exchanger.
[0006] Capillary electrophoresis is an analytical technique that
separates ions based on their electrophoretic mobility with the use
of an applied voltage. The electrophoretic mobility is dependent
upon the charge and the hydrodynamic radius of an ionic species. It
has been shown that CE separations can be carried out in short
fused silica capillaries in conjunction with mass spectrometry in
the migration time range of a few seconds [4].
[0007] Although two-dimensional systems were known, a combination
of ion exchange chromatography methods and electrophoresis based
methods was not contemplated in the past for various reasons. In
particular, the relatively slow separation speed of conventional CE
based on the use of long capillaries was deemed to exclude the
construction of a corresponding two-dimensional separation system.
A difference between both systems is the fundamentally different
flow characteristic that develops in the injection cell around the
CE separation capillary.
[0008] One crucial aspect for the technical realization of
two-dimensional separations with the conversion of all sample
components from the first to the second dimension of separation is
the use of a modulator which controls the transfer from the first
to the second dimension. In DE 19717738C1 [5] it has been found
that with the process of capillary batch injection analysis (CBIA)
small sample volumes in the nanoliter range, which are handled by
means of a capillary coupled to a microliter syringe, may be
injected directly onto the surface of a sensor in a detection cell
filled with electrolyte solution, and the small injected sample
volumes may then be dispersed in the electrolyte reservoir by a
stirrer, such that the measurements can be repeated at a high
frequency, showing only a negligible base line drift due to the
large dilution in the electrolyte reservoir. It has been shown
later that the CBIA concept can also be adapted as an injection
concept for CE [6]. However, both in the classical CBIA as well as
in the case of CBIA-CE, only discrete sample volumes can be taken
up by means of a capillary and then injected to a sensor surface or
to the inlet of a CE capillary, respectively.
[0009] Batch processing is labor-intensive and time consuming, as
for every batch, the system has to be cleaned and the solutions
have to be prepared before the system is ready for the next batch.
Furthermore, batch processing is more error-prone due to being
labor-intensive, and comparison of the results of the analysis of
the different batch samples can vary due to the practically
separate experimental setup conditions for every batch.
[0010] It was an objective of the present invention to provide a
device and a method for an improved separation of ionic species,
which has increased peak capacity, is more cost-effective, and less
error-prone than the methods known and used in conventional
manner.
SUMMARY OF THE PRESENT INVENTION
[0011] The present invention provides an improved method of
separating ionic species and a device for use of such a method.
[0012] It was surprisingly found that a highly efficient separation
of ionic species is possible by coupling ion chromatography (IC)
and capillary electrophoresis (CE) using a modulator as defined in
the claims. The IC.times.CE method of the present invention is a
two-dimensional separation system that allows a high orthogonality
for ion separation and represents a very attractive new technology
in the field of separation processes. It was found that a
combination of IC with retention times that are typically in the
range of 5-30 minutes, with a CE system is possible when using the
specific modulator providing a sequential injection of small volume
segments in the nanoliter range into the CE system at time
intervals of a few seconds.
[0013] The devices and methods of the present invention allow
continuous operation, i.e. IC can be coupled to CE on-line, which
means that both separation methods can be operated without
interruption of the basic setup. This was accomplished by the
modulators of the invention used to control the transfer of the IC
carrier flow to the CE system. The new system offers many
advantages and can be used to separate charged molecules, like
mixtures of nucleotides and cyclic nucleotides, in an efficient
manner.
[0014] The method of the present invention uses IC in the first
dimension and CE in the second dimension. Both methods per se are
well-known in the field and the known methods and devices can be
used in common manner and the optimal conditions can be found based
on the species to be analyzed.
[0015] The critical part of the method is the transfer of the IC
effluent to the CE system which is outlined below in detail.
[0016] Thus, a device of the present invention comprises an ion
chromatography (IC) system, a capillary electrophoresis (CE)
system, and a modulator.
[0017] The IC system can be a known system including a suppressor
which allows obtaining an effluent with a carrier that is highly
pure water. The system is operated continuously with retention
times normally being in the range of 5 to 30 minutes, depending on
the sample composition. In a preferred embodiment, the IC system is
a capillary IC system.
[0018] The second system is a CE system that comprises an
electrolyte vessel with electrophoresis buffer, and a high voltage
electrode. CE systems are known per se, for the present invention a
capillary CE system is used with a short capillary.
[0019] Both systems are connected via a transfer capillary which
provides for the transfer of the IC effluent to the CE system and
an injector which provides for the injection of volume segments of
the IC effluent into the CE capillary. The effluent is continuously
provided and transferred through the transfer capillary and volume
segments are injected in time increments, i.e. continuously in the
form of discrete increments. The injection of volume segments is an
essential part of the method of the present invention. A modulator
regulates the injection to avoid that in the immediate area around
inlet of the CE capillary a breakdown of the electrophoretic
current occurs. This can happen when the incoming IC carrier flow
from the outlet of the transfer capillary within the electrolyte
vessel filled with electrophoresis buffer is continuously very
close to the inlet, because the IC carrier flow exhibits very low
background conductivity. Without the electrophoretic current, the
CE could not be operated. Therefore, in order to maintain a
continuous carrier flow through the IC transfer capillary without
resulting in a breakdown of the electrophoretic current in the
electrolyte vessel, the modulator used for the IC.times.CE coupling
of the present invention is adapted, to manage sequential
injections of segments of the IC carrier flow from the outlet of
the IC transfer capillary into the inlet of the CE capillary. In
other words, it is essential that volume segments instead of a
continuous flow are injected into the CE capillary. This is
achieved by injector means providing for discrete volume parts of
effluent being injected.
[0020] The modulator can be any injector device that provides for
delivering discrete volume segments of effluent from the IC system
to the CE system. In the following, two preferred embodiments for a
modulator/injector are described in more detail, i.e. a guidance
and positioning system and a valve system. In a first alternative,
the distance between the outlet of the transfer capillary and the
inlet of the CE capillary is modified periodically, also defined as
cycling, preferably by a microprocessor-controlled guidance and
positioning system, which governs the movement of the outlet of the
transfer capillary towards and away from the inlet of the
separation capillary and/or the movement of inlet of the separation
capillary towards and away from the outlet of the transfer
capillary. In its initial position, the distance between outlet of
the transfer capillary and inlet of the separation capillary is too
large for hydrodynamic transfer of the liquid zone emerging from
the outlet of the transfer capillary to occur. In this condition IC
effluent enters the electrolyte solution without entering the CE
separation capillary. The movement of the outlet of the transfer
capillary towards the inlet of the separation capillary, or the
movement of the inlet of the separation capillary towards the
outlet of the transfer capillary, or the movement of both, reduces
the distance until defined hydrodynamic transfer between the
capillaries can occur. This transfer is called injection. At least
one of the capillaries is guided and positioned from a first
position to a second position to change the distance between outlet
of the transfer capillary and inlet of the separation capillary
such that in a first position, when both, outlet and inlet, are in
a distance from each other, an injection does not occur, and in a
second position, when both, outlet and inlet, are close to each
other, an injection occurs. Both capillaries can be arranged in
axial direction, sideways or in any other direction that allows for
guidance and positioning, preferably until they are in alignment.
Preferably, the capillaries are moved in axial direction. Either
the separation capillary or the transfer capillary can be in a
fixed position. Alternatively, both capillaries can be moved
towards and away from the other capillary. Preferably, the CE
separation capillary is in a fixed position and the transfer
capillary is moved in axial direction.
[0021] It has been found that a further advantage of the injection
by movement of the capillary/capillaries is obtained, i.e. that by
the movement, a convection in the electrophoresis buffer in the
electrolyte vessel is caused, which dilutes the emerging IC carrier
flow, and thereby minimizes the influence of the IC carrier flow on
the stability of the electrophoretic current. This allows using
this system without the need for stirring, such as a mechanical
stirrer in the electrolyte vessel. Therefore, although a stirrer
can be used for a device of the present invention, it is not
necessary and in one embodiment the CE system does not comprise a
stirrer.
[0022] In another embodiment, convection in the electrophoresis
buffer in the electrolyte vessel can be caused by a stirrer, which
can be operated continuously, or intermittently, e.g. only during
the interval between injection steps. The use of a stirrer can be
useful to reduce the distance between the outlet of the transfer
capillary and the inlet of the separation capillary during the
interval in between injection steps, as the emerging IC carrier
flow is rapidly diluted by the convection caused by the stirring.
As in the method of the present invention operation of a stirrer is
not essential because the movement of the capillary/capillaries is
causing sufficient convection for operation of the CE, but can be
still useful for some embodiments, the skilled artisan can choose
the optimal conditions, either with or without a separate
stirrer.
[0023] In a second alternative, a switching valve is used between
the outlet of the transfer capillary and the IC system. The valve
can be positioned near the outlet of the IC column, or near the
outlet of the transfer capillary. The opening and shutting of the
valve can be controlled by a microprocessor and is timed to inject
segments of IC carrier flow into the separation capillary. The part
of the carrier flow that is not used for CE separation can either
be discarded or can flow into the electrolyte vessel. In this case
the distance between the outlet of the transfer capillary and the
inlet of the separation capillary can be smaller and kept constant,
because between injection steps, no IC carrier flow is emerging and
affecting the integrity of the electrophoretic circuit.
[0024] The volume of the segments to be injected can be determined
in accordance with the conditions used, such as the CE system, the
sample composition etc. The skilled artisan can find the optimal
volume in routine experiments. Preferred ranges are described
below.
[0025] Other means for cycling or dosing volume segments, i.e.
providing discrete volume segments intermittently can be used.
[0026] The device of the present invention can additionally
comprise detection means for analysis of the IC and/or CE effluent.
Detectors for IC and CE systems are known in the art and can be
used for the present device accordingly. Preferably, a mass
spectrometer is used. Thus, in a preferred embodiment the outlet of
the CE capillary can be connected with a mass spectrometer for the
detection of the substance zones separated by IC.times.CE.
DESCRIPTION OF DRAWINGS
[0027] FIG. 1 shows the basic structure of a two-dimensional
separation system of the present invention for on-line coupling of
ion chromatography (IC) and capillary electrophoresis (CE) in
conjunction with mass spectrometric (MS) detection.
[0028] (A): microprocessor-based controller of the IC.times.CE
coupling with sequential upward and downward movement of the IC
transfer capillary (B), and switching function (On/Off) of the
stirrer (E); (B): IC transfer capillary; (C): CE separation
capillary; (D): platinum high voltage electrode for the CE
separation; (E): stirrer; IC system: ICS-5000 (Thermo); MS system:
micrOTOF-MS (Bruker Daltonics)
[0029] FIG. 2 shows the result of a two-dimensional separation
IC.times.CE of nucleotides and cyclic nucleotides as a contour plot
(a) and as a combined chromatoelectropherogram (b); substances:
AMP, GMP, CMP (each 300 .mu.M), and cAMP, cGMP, cCMP (each 100
.mu.M); separation conditions IC: lonSwift MAX-200 anion column;
eluent: 40 mM KOH; injection volume: 0.4 .mu.l; flow rate; 5
.mu.L/min; transfer capillary: 60 cm in length/75 .mu.m inner
diameter (ID); CE separation conditions: separation buffer: 25 mM
NH.sub.4Ac/NH.sub.3 pH 9.15; capillary dimensions: 20.5 cm in
length/25 .mu.m inner diameter; separation voltage: 22.5 kV;
injection time: 2 seconds each; interval between two consecutive
injections: 17 seconds.
[0030] FIG. 3 shows an illustration of the modulation process
(movement of the transfer capillary towards the separation
capillary). One injection interval (I) comprises injection time
t.sub.inj, pre-injection time t .sub.preinj and the time the
stepper motor needs to switch between them.
DEFINITIONS
[0031] Ion chromatography (most commonly ion-exchange
chromatography) is a process that allows the separation of ions and
charged polar molecules based on their affinity to the ion
exchanger. It can be used for almost any kind of charged species or
molecule that can form ionic species, any molecule that can be
protonated or deprotonated. Examples of suitable substances include
inorganic compounds, like salts, acids and bases, or organic
molecules, like large proteins, small nucleotides and amino acids.
Typically, the sample is loaded onto the column in the form of an
aqueous solution and an eluent, i.e. an aqueous solution with
suitable eluting power, known as the mobile phase, is used to carry
the sample through the column comprising the stationary phase. The
stationary phase is typically a resin or gel matrix consisting of
agarose or cellulose beads with covalently bonded charged
functional groups. The target analytes (anions or cations) are
retained on the stationary phase but can be eluted by increasing
the concentration of a similarly charged species that will displace
the analyte ions from the stationary phase. For example, in anion
exchange chromatography, the negatively charged analyte could be
displaced by a rather high hydroxide ion concentration in the
mobile phase. The analytes of interest can be detected by detector
means, typically by using their conductivity or UV/Visible light
absorbance for detection.
[0032] Capillary electrophoresis (CE) is an analytical technique
that separates ions based on their electrophoretic mobility with
the use of an applied high voltage. The electrophoretic mobility is
dependent upon the charge of the ion and the hydrodynamic radius.
The rate at which the ion moves is directly proportional to the
applied electric field--the greater the field strength, the faster
the speed of migration. Neutral species have no specific mobility
but are transported by the so-called electro-osmotic flow. If two
ions are the same size, the one with greater charge will exhibit
higher electrophoretic mobility. For ions of the same charge, the
smaller ion has less friction and overall higher electrophoretic
mobility. Capillary electrophoresis is attractive because it
provides high separation efficiency.
[0033] "Suppression" or "suppressor" is used for IC to increase
analyte signal in case of conductivity detection. The background
conductivity of the chemicals used to elute analyte species from
the ion-exchange column is reduced. This improves the conductivity
measurement of the ions being tested. When using IC with
suppression the IC carrier flow or effluent has low background
conductivity, corresponding to pure or ultrapure water. The use of
suppressors in IC is well-known and the skilled person can find
suitable ones easily. An optimal suppressor is one that provides an
effluent with a background conductivity as low as possible.
[0034] The term "continuous" when used in the present description
refers to a continuous operation in contrast to a batchwise
operation. In particular it refers to a method where the IC carrier
flow or effluent is continuously transferred from the IC system
through the transfer capillary to the injector, where it is
injected on-line, without interrupting the operation of the IC or
CE system, into the CE capillary. This does not exclude that the
flow can encompass short interruptions, e.g. by shutting off a
valve in the modulator between the IC system and the CE system.
[0035] "On-line coupling" means that the two separation methods are
coupled in a way that allows continuous separation and detection of
the ionic species via both separation techniques in sequence.
[0036] "Injection" means the transfer of a discrete volume segment
of the IC carrier flow or effluent from the outlet of the transfer
capillary to the inlet of the separation capillary and the entrance
into the separation capillary. One interval of injection is
comprising the injection time t.sub.inj, the preinjection time
t.sub.preinj, and the time the positioning unit of the modulator
needs to move the capillary from injection position to preinjection
position and backwards (see also FIG. 3).
[0037] "Nanoliter volume" means volumes of less than 1 .mu.l.
[0038] "Volume segments" are discrete volumes of the effluent that
are created by injector means, for example by modifying the
distance between transfer and separation capillary or by a
valve.
[0039] "Ionic species" means ions or molecules that can be charged.
Examples are substances that dissociate or can be protonated or
deprotonated in solution, such as organic molecules like amino
acids, peptides and proteins, nucleotides, or inorganic compounds
like acids, bases, salts etc..
[0040] "Time increment" means a predetermined time period, in
particular a time period that is used in the cycling mode.
[0041] "Effluent" or "IC carrier flow" is used interchangeably and
defines the mobile phase that flows out of the IC column or
capillary, respectively.
[0042] "Sample" is a composition that shall be analyzed and usually
is an aqueous solution of analytes.
[0043] "Analyte" can be any substance that can be analyzed with an
IC.times.CE system, such as anions and cations.
[0044] The term "cycling" refers to a mode of injection that is
preferably used in the method of the present invention, where
repeatedly volume segments are provided and injected
intermittently.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention provides devices and a method for
two-dimensional IC.times.CE separation of ionic species, wherein
the two separation methods are coupled on-line, and with continuous
operation.
[0046] The device comprises an ion chromatography (IC) system,
which comprises a suppressor. By using a suppressor, the effluent
obtained from the IC column comprises a solution having low
background conductivity with the analyte species separated therein.
This causes a so-called "stacking" effect during the sequential
injection into the CE system, due to the differences in
conductivity of the electrophoresis buffer and injected sample
solution, which result in a sharpening of the injected bands and in
a signal amplification. IC systems are known and the known systems
can be used for the device and the method of the present
invention.
[0047] With an IC.times.CE system of the present invention, wherein
the analyte zones separated by IC are present in a carrier flow of
solution having low background conductivity after suppression, it
is possible to apply a short injection interval of less than 10 s,
preferably of less than 2 s, in order to keep effects on the
electrophoretic current low and to apply narrow injection segments.
Preferably the IC system is a capillary IC system.
[0048] The IC carrier flow is transferred via a transfer capillary
either partially or completely, into an electrolyte vessel
comprising an electrophoresis buffer, and a high voltage electrode,
preferably a platinum electrode. CE systems with short separation
capillaries (<50 cm) can be used. In a typical CE system an
electric field is maintained permanently during the operation of
the IC.times.CE via the electrode and the electrophoresis buffer.
This voltage is about 1-100 kV; preferably it is about 10 to 35,
more preferably 15 to 30 kV. The field is maintained between the
electrode and the grounded interface. Usually the outlet of the
electrophoresis buffer filled CE separation capillary is connected
with a detector, preferably a mass spectrometer.
[0049] The electrolyte vessel comprises an electrophoresis buffer,
which has a volume which is significantly higher than the volume of
the volume segments emerging from the outlet of the transfer
capillary between injection steps. For example, the volume segments
being in the nanoliter range, the volume of the electrophoresis
buffer can be in the range of 1 to 10 ml, such as about 2 ml. In
this way, it is ensured that the influence of the IC carrier flow
on the stability of the electrophoretic current is ensured. This
influence can be further minimized by causing a convection in the
buffer, for example by movement of the capillary and/or by the
operation of a stirrer. The optimal conditions can be selected by
the skilled person depending on the size of the electrolyte vessel
and the selected flow rate of the IC carrier flow. The CE buffer
should be replaced in the electrolyte vessel at appropriate
intervals to ensure stable electrophoretic separations as it is
known to the skilled person. It has been found that an IC carrier
flow rate less than 10 .mu.l/min is preferable as it leads to good
results. With higher flow rates the effect of the IC carrier flow
on the integrity of the electrophoretic circuit could be
compromising for the stability of the CE. A flow rate in the range
of 1 to 8, such as about 5 .mu.l/min is preferred (see also Example
3). Higher flow rates until up to 10 .mu.l/min can be applied.
Potential detrimental effects on the stability of the
electrophoretic current can then be compensated by causing
convection in the electrophoresis buffer to rapidly dilute the IC
carrier flow. This can for example be achieved by a switchable
stirrer, which is optionally switched on during the interval
between injection steps, or by other known devices creating
convection.
[0050] To avoid any interference, the transfer capillary connecting
the IC system with the electrolyte vessel should be made of
nonconductive material, such as fused silica or plastic; preferably
it is made of fused silica. The separation capillary is also
preferably made of fused silica. The fused silica capillaries can
be coated with polyimide. The polyimide coating can then be removed
at the ends of the capillary prior to use. For example, the
polyimide coating can be removed at the end of the capillary at a
length of about 5 mm. Furthermore, prior to use, both ends of the
separation capillary and the injection end of the transfer
capillary can be polished with polishing papers, preferably with a
grit size of 32 .mu.m and 12 .mu.m, preferably at an angle of about
90.degree., to smoothen the surface. Furthermore, prior to use, the
separation capillary can be flushed to condition the capillary. For
example the separation capillary can be flushed sequentially with
100 mM NaOH, preferably for about 10 minutes, with ultrapure water
from a Milli-Q system, preferably for about 10 minutes, and with
the background electrolyte, preferably for about 30 minutes. The
background electrolyte can consist of a 25 mM ammonium acetate
buffer adjusted with ammonia to pH=9.15. The buffers used in the
application are preferably filtered prior to use, for example by a
syringe filter (0.2 .mu.m). Moreover, the size of the transfer
capillary should be adapted to the volume to be used and to the
size of IC column and CE capillary. Preferably, the transfer
capillary has an inner diameter of less than 500 .mu.m; preferably
the inner diameter is in the range of 150 to 50 .mu.m, such as
about 75 .mu.m.
[0051] The essential part of the device of the present invention is
a modulator, which controls the transfer of IC carrier flow from
the IC transfer capillary into the CE system. The modulator
comprises a transfer capillary and injector means.
[0052] Injector means, optionally regulated or controlled by a
microprocessor, provide for the creation of volume segments that
are injected in time increments rather than continuously. Any
device that can provide for intermittent delivery of volume
segments of the effluent from the transfer capillary can be used.
In preferred embodiments either a positioning and guidance system
for modifying the distance between transfer capillary and
separation capillary or a valve system are used. In one embodiment
the injection, the time increments and the volume segments,
respectively, are controlled by adjusting the distance between the
outlet of the transfer capillary and inlet of the separation
capillary regularly, or by opening and shutting a switching valve
in the transfer capillary. Distance adjustment can be achieved by
movement of one or both of the capillaries towards and away from
the other capillary. This movement is controlled by a positioning
and guidance system, optionally controlled by a microprocessor.
Transfer of IC carrier flow into the CE system can occur, when the
distance between the openings of the capillaries is small enough to
allow transfer of a defined volume segment of IC carrier flow by
hydrodynamic force.
[0053] In a preferred embodiment, the modulator comprises a
microprocessor, which, in the first alternative, controls the
movement of the transfer capillary towards and away from the inlet
of the CE separation capillary. Every few seconds small volume
segments are sequentially injected into the inlet of the separation
capillary by the microprocessor-controlled movement of the IC
transfer capillary, for example in axial direction (see B in FIG.
1). The movement starts from an initial or first position of the
transfer capillary, wherein the distance between outlet of the
transfer capillary and inlet of the separation capillary is
selected so that the liquid zone emerging from the outlet of the
transfer capillary does not enter the inlet of the CE capillary
hydrodynamically. This distance is reduced to a smaller distance
between the outlet of the transfer capillary and the inlet of the
separation capillary, or even until direct contact is achieved,
i.e. to the second position. At this smaller distance, a defined
hydrodynamic transfer of the liquid zone into the inlet of the CE
capillary can occur. Suitable distances depend on the overall
hydrodynamic situation in the electrolyte vessel, which depend e.g.
on the flow rate through the transfer capillary, the amount of
stirring and/or convection in the electrolyte vessel, or the flow
velocity in the CE system. The flow rate should be selected such
that between injection steps, excessive IC carrier flow into the
electrophoresis buffer is avoided. In this way, less IC carrier
flow comprising the analyte is lost, and the electrophoretic
current is less affected.
[0054] In a preferred embodiment, the modulator is a modified
capillary batch injection (CBI) device. Briefly, the modulator
consists of a vertical positioning unit moving the transfer
capillary, which is fixed on a holder, up and down in axial
direction (see arrow in detailed part of FIG. 1 showing the
electrolyte vessel). This can be achieved by a 1.8.degree. stepper
motor with leadscrew which reaches a positioning precision of 1
.mu.m/step. The end of the transfer capillary can be guided through
a 0.38 mm inner diameter glass guide (Hilgenberg, Malsfeld,
Germany), fixed in a purpose-built manual x,y-positioner, into the
electrophoretic vessel. With help of the positioning unit the
transfer capillary is aligned with the separation capillary, which
is located in axial direction on the bottom of the electrophoretic
vessel. Positioning can be controlled using a laboratory-modified
microscopic video camera (e.g. DigiMicro 1.3, dnt, Dietzenbach,
Germany). The electrophoretic vessel cell can be further equipped
with a stirrer. The rate of injection steps or the timely sequence
of volume segments depends on the separation speed of the CE
system. All components of the first volume segment injected into
the CE separation capillary preferably have already reached the
detector, before the next volume segment is injected. In this way
it is ensured that all signals detected can be unambiguously
assigned to the respective volume segment injected into the CE.
Basically, it is ensured that ionic species of a subsequent
injected volume segment cannot overtake ionic species of the
previously injected volume segment in the CE separation capillary.
The interval in between injection steps depends, inter alia, on the
length of the CE separation capillary used. The shorter the column,
the shorter the interval in between injections steps can be. In an
example that is described later, the CE separation capillary was
20.5 cm in length, which allowed an interval of about 17 seconds.
This interval comprises the injection time t.sub.inj of 2 s and the
pre-injection time t.sub.preinj of 15 seconds. In the case of
co-electroosmotically migrating species shorter preinjection
intervals of less than 10 s can be possible.
[0055] The exact distances between the outlet of the transfer
capillary and the inlet of the separation capillary must be adapted
to the experimental conditions, which can be done by the skilled
person. A typical flow rate for a capillary IC system is less than
10 .mu.l/min, preferably about 1 to about 8, such as about 5
.mu.l/min. With a higher flow rate, there is a risk that the volume
of IC carrier flow is not injected into the CE separation
capillary, leading to a dilution of the electrophoresis buffer.
This could affect the stability of the electrophoretic current
necessary for the operation of the CE. In one embodiment a cycling
between a distance of less than 100 .mu.m for injection, and more
than 150 .mu.m in the intermittent period, is used. In other words,
during injection mode a typical distance between outlet of the
transfer capillary and the inlet of the separation capillary is
less than 100 .mu.m. The distance between the injection cycles is
typically increased to greater than about 150 .mu.m. If a stirrer
is used, the suitable distances can be reduced. For example, the
distance during injection mode can be from about 30 to about 50
.mu.m.
[0056] The injection time, governed by either the length of time in
which the capillaries are positioned in injection mode (short
distance), or by the time in which the switching valve is open, can
be up to 10 seconds. Preferably, the injection time is about 2
seconds.
[0057] After injection, the transfer capillary or the separation
capillary, or both, is/are moved back to the initial position. The
rapid homogeneous distribution of liquid, which has emerged from
the outlet of the transfer capillary, in the electrolyte vessel but
has not been injected into the separation capillary, can be
supported by switching on a stirrer (see E in FIG. 1). However, the
use of a stirrer is optional. The results shown in FIG. 2 were
obtained without stirring. In addition to the mass spectrometric
detection, which generates the detection signals for the
IC.times.CE separation, also the conductivity detection of the IC
can be recorded and can be used in addition for the analytical
evaluation.
[0058] In another preferred embodiment, the capillaries are both in
a fixed position, and the modulator is associated with a switching
valve which controls the IC carrier flow through the transfer
capillary by opening and shutting the valve. The switching valve
can be positioned near or at the inlet of the transfer capillary,
or near or at the outlet of the transfer capillary, or anywhere in
between. In other words, the valve can withdraw small volumes of
effluent directly at the outlet of the IC column and deliver those
volume segments via the transfer capillary to the CE capillary,
where the outlet of the transfer capillary is in a position to
allow injection into the CE capillary. In another embodiment, the
effluent of the IC column is transferred completely or partially to
the transfer capillary and the valve being positioned near or at
the outlet of the transfer capillary provides for the delivery of
suitable volume segments to the inlet of the CE capillary. The
switching valve can be opened and shut in a controlled manner,
optionally controlled by a microprocessor, leading to injection
intervals corresponding to the injection intervals achieved by the
sequential movement described for the first alternative of the
modulator above. During the time period, wherein the switching
valve is open, defined liquid segments are transferred
hydrodynamically from the outlet of the transfer capillary to the
inlet of the CE separation capillary. One advantage of this
alternative solution is that only in the injection interval carrier
liquid is introduced into the CE system. This embodiment is
particularly useful for those IC systems where a high carrier flow
is obtained.
[0059] The devices of the present invention further comprise a
capillary electrophoresis (CE) separation capillary. To obtain
optimal results, the separation capillary should be as short as
possible. It has been found, that a separation capillary having a
length of less than 50 cm can be used, preferably the capillary has
an inner diameter of less than 100 .mu.m. Preferably, the
separation capillary is less than 35 cm, more preferably less than
25 cm in length, and has an inner diameter in the range of about 35
to about 20, such as about 25 .mu.m. The inlet of the separation
capillary, in one embodiment, is essentially in axial alignment
with the outlet of the transfer capillary.
[0060] Preferably, the devices of the present invention further
comprise an interface connecting the outlet of the separation
capillary with a detector. For example, the short CE separation
capillary, which is filled with electrophoresis buffer, can be
coupled at its outlet to a commercial "sheath flow"--electrospray
ionization (ESI) interface for combination with a mass
spectrometer. In the electrolyte vessel, a high voltage between the
electrophoresis buffer and the ESI interface ground can be
maintained permanently via a platinum electrode placed in the
electrolyte vessel (see D in FIG. 1).
[0061] The device of the present invention can be used in a method
of the present invention for two-dimensional separation of ionic
species by on-line coupling of ion chromatography and capillary
electrophoresis (IC.times.CE), comprising the sequential injection
of volume segments of the IC carrier flow into the CE system
[0062] The method of the present invention for two-dimensional
separation of ionic species by online coupling of ion
chromatography (IC) and capillary electrophoresis (CE), comprises
the following steps:
[0063] a) injecting a sample into an IC system comprising a
suppressor;
[0064] b) transferring IC effluent through a transfer capillary to
a CE system comprising an electrolyte vessel with electrophoresis
buffer, a separation capillary and a high voltage electrode;
[0065] c) injecting volume segments of effluent into a separation
capillary of the CE system via injector means.
[0066] In a first step, a sample is loaded onto an IC system. This
IC system is preferably a capillary IC system. The sample can be
any compound or mixture of compounds, which comprises ions under
operating conditions. For example, the sample can comprise mixtures
of amino acids, or nucleotides, or cyclic nucleotides or any other
substance that can form ionic species as a result of dissociation
or protonation/deprotonation as defined above. In a second step,
the ionic species in the sample are separated in the IC system and
suppressed. After suppressing the ions, the analyte is present in
solution, usually aqueous solution with low background
conductivity, for example in highly pure water. The solution
containing the analyte zones is the IC carrier flow or effluent,
which is then transferred via and through a transfer capillary as
defined above to the outlet of the transfer capillary, which ends
in the electrophoresis buffer comprised in an electrolyte vessel as
defined above.
[0067] In a third step, volume segments of IC carrier flow or
effluent are injected into the inlet of the separation capillary
via injection means, as defined above.
[0068] If the sample containing ionic species to be separated
comprises a mixture of amino acids, the separation conditions are
preferably selected so that the amino acids are present in the
sample as anionic species during separation by IC. This can be
achieved by using an alkaline IC buffer. The separation conditions
should then be selected so that the amino acids separated by IC and
injected into the separation capillary are present as cationic
species during separation by CE. This can be achieved by using an
acidic CE buffer such as formiate. A formiate buffer is also
compatible with the subsequent detection technique such as mass
spectrometry. By switching from anionic species to cationic species
between the two combined separation techniques by selecting
alkaline and acidic buffers, the efficiency of the combined
separation techniques is increased due to the difference in
selectivity caused by the different states of charge. Furthermore,
the injection frequency can be increased because CE separation can
be achieved more rapidly with cationic analytes compared to anionic
analytes.
[0069] In one alternative, the transfer is controlled by movement
of one or both of the capillaries towards and away from each other,
for example in their axial direction. As soon as the distance
between the openings of both capillaries is small enough to allow
hydrodynamic transfer of a defined volume of IC carrier flow,
injection occurs. For example, if the separation capillary is in a
fixed position, and the modulator comprised a positioning and
guidance system for the movement of the transfer capillary, the
transfer capillary is then moved to set a distance of less than 100
.mu.m between the outlet of the transfer capillary and the inlet of
a separation capillary of a CE system. This short distance is then
kept for less than 10 seconds. During this time period, which is
also termed injection period, a defined volume segment is
introduced/injected into the inlet of the separation capillary of
the CE system. After the injection step the transfer capillary is
moved to increase the distance between the outlet of the transfer
capillary and the inlet of the separation capillary of the CE
system to about more than 150 .mu.m. During these steps, the IC
carrier flow is continuous.
[0070] The CE carrier flow can then be transferred to a detector
via an interface as described above. For example, the separation
capillary, which is filled with electrophoresis buffer, can be
coupled at its outlet to a commercial "sheath flow"--electrospray
ionization (ESI) interface for coupling to a mass spectrometer. The
injection steps can be repeated until the IC carrier flow is
processed to the latest zone eluting from the IC.
[0071] During the method usually a high voltage of about 1-100 kV,
preferably of about 20 kV, is maintained between the electrode in
the electrolyte vessel and the interface between the separation
capillary outlet and the detector.
[0072] FIG. 2 illustrates the result of an IC.times.CE separation
with mass spectrometric detection of a mixture of nucleotides and
cyclic nucleotides. The graph shows the intensities of the
extracted mass spectrometric signals according to the retention
time of the IC and the migration time of the CE. With this
relatively simple model mixture of six components, the significant
increase in the peak capacity by the realized IC.times.CE
separation is illustrated. While using IC some nearly co-eluting
bands with base peak widths of 1-1.5 min can not be separated in
the first dimension, the CE in several cases allows a separation
with high separation efficiency (base peak widths of a few
seconds). In FIG. 2 it can be seen that the substances cCMP, CMP,
AMP and cAMP are only partially or not at all separated in the
first separation dimension (IC). The IC.times.CE, however, leads to
a complete separation of all model compounds and allows a
significant increase in peak capacity due to the high separation
efficiency of CE and due to the orthogonality of IC and CE. This
shows the superiority of the online coupled IC.times.CE method of
the present invention.
EXAMPLES
Example 1
[0073] A sample comprising nucleotides and cyclic nucleotides was
subjected to a two-dimensional separation using ion chromatography
(IC) and capillary electrophoresis (CE). The IC comprised a
suppressor to provide effluent comprising the analytes in highly
pure water.
[0074] The sample was injected into the IC system and continuously
eluted. The IC effluent was transferred through a non-conductive
transfer fused silica capillary to an electrolyte vessel of a CE
system containing electrophoresis buffer. Nanoliter volume segments
of the effluent were injected into the separation capillary of the
CE system by continuously modifying the distance between transfer
capillary and separation capillary. The distance between the outlet
of the transfer capillary and the inlet of the separation
capillary, which were in axial alignment, for injecting a nanoliter
volume segment was moved such that the outlet of the transfer
capillary had a distance of 50 .mu.m to the inlet of the separation
capillary. The short distance was kept for 2 seconds such that a
volume segment could enter the inlet of the separation capillary of
the CE system and then the transfer capillary was withdrawn in
axial direction to increase the distance to 300 .mu.m. This
distance was maintained for 15 seconds, before the next injection
was done. The movement of the transfer capillary was controlled by
a modulator comprising a positioning and guidance system controlled
by a microprocessor. A high voltage of about 20 to 25 kV was
maintained between the electrode in the electrolyte vessel and the
interface between the separation capillary outlet and the
detector.
[0075] For IC the following separation conditions were used:
IonSwift MAX-200 anion column; eluent: 40 mM KOH; injection volume:
0.4 .mu.l; flow rate; 5 .mu.L/min; transfer capillary: 60 cm in
length/75 .mu.m inner diameter (ID)
[0076] For the CE separation the following conditions were used:
electrophoresis buffer: 25 mM NH.sub.4Ac/NH.sub.3 pH 9.15;
capillary dimensions: 20.5 cm in length/25 .mu.m inner diameter;
separation voltage: 22.5 kV; injection time: 2 seconds each;
interval between two consecutive injections: 17 seconds.
[0077] The results are shown in FIGS. 2a and 2b.
[0078] FIG. 2a shows the result of the two-dimensional separation
IC.times.CE of nucleotides and cyclic nucleotides as a contour
plot.
[0079] FIG. 2b shows in a combined chromatoelectropherogram that
nucleotides and cyclic nucleotides could be resolved by using the
method of the present invention. As can be seen the peaks for AMP,
GMP, CMP (each 300 .mu.M), and cAMP, cGMP, cCMP (each 100 .mu.M)
are clearly separated.
Example 2
[0080] A further sample comprising nucleotides and cyclic
nucleotides was subjected to a two-dimensional separation using ion
chromatography (IC) and capillary electrophoresis (CE) as described
above in Example 1 with the following additional details.
[0081] Prior to using fused silica capillaries (Polymicro
Technologies, Phoenix, Ariz., USA) in the separation techniques the
capillaries were prepared and conditioned as follows. Both ends of
the separation capillary (length=20.5 cm, inner diameter=25 .mu.m)
and the injection end of the transfer capillary (length=60 cm,
inner diameter=75 .mu.m) were polished with polishing papers (32
.mu.m and 12 .mu.m grit size) at a 90.degree. angle until the
surface was smooth. The polyimide coating was removed at a length
of about 5 mm. Before each use, the separation capillary was
flushed 10 minutes with 100 mM NaOH, 10 minutes with ultrapure
water from a Milli-Q system, and 30 minutes with the background
electrolyte consisting of a 25 mM ammonium acetate buffer adjusted
with ammonia to pH=9.15. The buffer was filtrated before use with a
syringe filter (0.2 .mu.m) (Carl-Roth, Karlsruhe, Germany). The
measurements were performed applying a separation voltage of 22.5
kV.
[0082] An ICS-5000 (Dionex, Thermo Scientific) ion chromatograph
was used for capillary-scale IC separations. It consisted of a dual
pump module with both capillary (pump 1) and analytical pump (pump
2), an eluent generator module (EG KOH 300 with subsequent trap
column), and a detector/chromatography module. The latter module
comprises an in-line eluent degasser, a four-port injection valve
(injection volume, 0.4 .mu.L), a column oven, an anion capillary
eluent suppressor, and a conductivity detector.
[0083] The capillary high performance (cHPIC)
detector/chromatography module was thermally controlled at
10.degree. C. Dionex Ion-Swift MAX-200 column (0.25.times.250 mm)
with appropriate guard column (0.25.times.50 mm), both operated at
35.degree. C., were used for anionic separation.
[0084] Instrument control and data acquisition were performed using
Chromeleon 6.8 software. The eluent concentration (KOH) was kept
constant at 40 mM hydroxide during a run.
[0085] A modified capillary batch injection (CBI) device was used
as modulator to control the transfer of the IC effluent by a
movable transfer capillary (FIG. 1B) into the CE separation
capillary. The modulator consists of a vertical positioning unit
moving the transfer capillary, which is fixed on a holder, up and
down in axial (z) direction (see arrow in detailed part of FIG. 1
showing the electrolyte vessel). This is achieved by a 1.8.degree.
stepper motor with leadscrew which reaches a positioning precision
of 1 .mu.m/step. The end of the transfer capillary is guided
through a 0.38 mm inner diameter glass guide (Hilgenberg, Malsfeld,
Germany), fixed in a purpose-built manual x,y-positioner, into the
electrophoretic vessel. With help of the positioning units the
transfer capillary is aligned with the separation capillary, which
is located in axial direction on the bottom of the electrophoretic
vessel. Positioning is controlled using a laboratory-modified
microscopic video camera (DigiMicro 1.3, dnt, Dietzenbach,
Germany).
[0086] A micrOTOF-MS (Bruker Daltonik, Massachusetts, USA) with a
coaxial sheath liquid electrospray interface (Agilent Technologies,
California, USA) was used for detection. A mixture of 2-propanol,
water, and ammonia (49.9: 49.9: 0.2, v/v/v) was used as sheath
liquid at a flow rate of 8 .mu.l/min. Nebulizer gas pressure was
set to 1 bar. The electrospray voltage was 4 kV.
Example 3
[0087] Modulation times of the IC.times.CE-MS measurements of a
model system comprising a mixture of nucleotides (AMP, GMP, CMP)
and cyclic nucleotides (cAMP, cGMP, cCMP) were studied and
optimized as illustrated in FIG. 3. These experiments were
performed without IC. Instead, a syringe pump (UMP3, WPI, Florida,
USA), a microliter syringe (1000 .mu.l Nanofil syringe, WPI), and a
fused silica injection capillary (inner diameter=75 .mu.m) were
used. The injection capillary was axially aligned with the
separation capillary and moved periodically up and down from
injection to preinjection positions while the sample was expelled
continuously with a flow rate of 5 .mu.l/min. The syringe was
filled with a model solution containing 150 .mu.M AMP and 50 .mu.M
cAMP in pure water. The CE high voltage source was permanently set
to 22.5 kV. To optimize the injection times, measurements were
performed with injection times of 1, 2, 4, and 8 seconds whereas
the preinjection time was kept at 15 seconds. Furthermore, the
preinjection time was varied (60, 30, 20, 15, 10, 6, and 2 seconds)
using an injection time of 2 seconds in order to optimize the
preinjection time. The shortest preinjection time possible without
overloading the capillary was 15 seconds. A further reduction of
preinjection times resulted in double peaks and separation was no
longer accomplished. 2 seconds were chosen as smallest injection
interval possible for this model system being a compromise between
small injection intervals and still sufficient signal
intensities.
Example 4
[0088] The IC flow rate was optimized by performing IC.times.CE-MS
measurements (AMP, GMP, CMP, 300 .mu.M each; cAMP, cGMP, cCMP, 100
.mu.M each) at different flow rates (2, 5, and 8 .mu.l/min). Low
flow rates led to peak broadening. Higher flow rates resulted in a
better IC separation efficiency. However, the flow rate is one of
the determining factors for the amount of substance injected into
the CE capillary. The IC effluent mainly consisted of pure water.
Thus, the higher the flow rate the more water was injected into the
capillary. Each injection led to a reduction of electrophoretic
current, which was recovering during the preinjection time. The
drop of current increased with higher flow rates. The
electrophoretic current dropped significantly (to 1-2 .mu.A) or
broke down completely with flow rates higher than 8 .mu.l/min. This
rendered any effort for separation impossible. Thus, 5 .mu.l/min
were chosen for all following measurements. At this flow rate
current fluctuations were in an acceptable range with an
electrophoretic current of 5.5-7 .mu.A.
Example 5
[0089] Furthermore, the effect of sample stacking in
cHPIC.times.CE-MS measurements was investigated. Sample stacking in
the CE separation capillary occurs when the specific conductivity
of the back-ground electrolyte is higher than the specific
conductivity of the sample plug. In the IC.times.CE-MS setup the IC
effluent, which was injected into the CE, consisted of analyte
zones in pure water due to the suppressor. The effect of stacking
during IC.times.CE-MS measurements was examined by means of a setup
comparable to IC.times.CE-MS where the IC was replaced by a
microsyringe pump. Mixtures of cAMP and AMP (50 .mu.M cAMP, 150
.mu.M AMP) dissolved in water or in background electrolyte were
used as samples. The respective sample was filled into the syringe
and the flow rate of the pump was set to 5 .mu.l/min. For the
measurements the high voltage source was permanently set to 22.5
kV. The optimized modulation times as described in Example 3 were
used. Before the first injection the capillary was kept 10 seconds
in preinjection position to equilibrate the flow of the pump. Then,
5 intervals of injection were performed and the electropherograms
were compared. The separation efficiency and peak heights of
analytes were significantly enhanced in case of the sample
dissolved in water compared to the sample prepared in background
electrolyte as a result of the stacking effect.
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